The Introduction Of Small Molecule Covalent Cross-linking Promoted The Assembly Of Peptides And Collagen

Supramolecular biomaterials with peptides and proteins as assembly modules are important functional materials in biomedicine and other fields due to their good biocompatibility and biological functionality.Among them,the coiled-coil has high sequence designability and structural stability,and is often used as a building block of high-order nanostructured materials,while collagen is the main component of extracellular matrix,and recombinant collagen material has a wide application prospect in tissue engineering and other fields.However,the development of a moderate,controllable and stable cross-linking method to promote module assembly is still a difficult point in the preparation of biomaterials.In this study,cysteine was inserted into the coiled-coil and recombinant collagen sequences through sequence design,and the spontaneous cross-linking of peptides and collagen was promoted by the introduction of bis(bromoethyl)benzene cross-linkers.They further assembled to form higher-order nanomaterials,which provided a new idea for the preparation of biomaterials.The main research content of this paper is divided into the following three parts:(1)The design strategy that introduced small molecules to form covalent bonds to assist cross-linking peptides and collagen.Through sequence design,cysteine was inserted at the end of the coiled-coil and recombinant collagen sequence,and bis(bromoethyl)benzene crosslinker was added to assist the spontaneous cross-linking of peptides and recombinant collagen under mild physiological conditions through thioether covalent bonds,promoting the polymerization assembly of peptides and recombinant collagen.(2)The introduction of cross-linkers promoted the cross-linking of peptides to form flexible long fibers.The coiled-coil E3/K3 was used as a template to explore the cross-linking conditions,and cysteine was introduced at the N-terminus of E3 and the C-terminus of K3,and CE3/K3 C was obtained by chemical synthesis.The cross-linker named 1,4-bis(bromomethyl)benzene was introduced,and the short peptide was able to form flexible long fibers about 100 nm long by cross-linking assembly within 48 h,and the length and number of long fibers increased over time,and the molecular particle size reached more than 1 μm in 144 h.At the same time,the effects of different substituent positions of cross-linkers on peptide polymerization efficiency and fiber morphology were investigated.The results showed that 1,2-bis(bromomethyl)benzene and 1,3-bis(bromomethyl)benzene could also react with cysteine to form thioether bonds,but the reaction efficiency and polymer stability may be reduced due to steric hindrance of the substituent position.Finally,the biosafety of the introduction of crosslinkers was verified by cytotoxicity experiments.(3)The introduction of cross-linkers promoted the cross-linking of recombinant collagen at the micron scale to form soft gels.The V-HC1 B collagen sequence designed with the human type I collagen α1 chain sequence as the template was selected,and cysteine was inserted at the C terminal or both ends of the sequence collagen domain,then V-B-CC was obtained by recombinant expression.Characterized by circular dichroism,collagen was able to fold correctly to form a three-strand helix structure.Finally,by introducing a 1,4-bis(bromomethyl)benzene cross-linker,the recombinant collagen could be assembled to form soft gels.